Characterization of Pseudomonas spp. contamination and in situ spoilage potential in pasteurized milk production process.

To investigate the prevalence of Pseudomonas in the pasteurized milk production process and its effect on milk quality, 106 strains of Pseudomonas were isolated from the pasteurized milk production process of a milk production plant in Shaanxi Province, China. The protease, lipase and biofilm-producing capacities of the 106 Pseudomonas strains were evaluated, and the spoilage enzyme activities of their metabolites were assessed by simulating temperature incubation in the refrigerated (7 °C) and transport environment (25 °C) segments and thermal treatments of pasteurization (75 °C, 5 min) and ultra-high temperature sterilization (121 °C, 15 s). A phylogenetic tree was drawn based on 16S rDNA gene sequencing and the top 5 strains were selected as representative strains to identify their in situ spoilage potential by examining their growth potential and ability to hydrolyze proteins and lipids in milk using growth curves, pH, whiteness, Zeta-potential, lipid oxidation, SDS-PAGE and volatile flavor compounds. The results showed that half and more of the isolated Pseudomonas had spoilage enzyme production and biofilm capacity, and the spoilage enzyme activity of metabolites was affected by the culture temperature and sterilization method, but ultra-high temperature sterilization could not completely eliminate the enzyme activity. The growth of Pseudomonas lundensis and Pseudomonas qingdaonensis was less affected by temperature and time, and the hydrolytic capacity of extracellular protease and lipase secreted by Pseudomonas lurida was the strongest, which had the greatest effect on milk quality. Therefore, it is crucial to identify the key contamination links of Pseudomonas, the main bacteria responsible for milk spoilage, and the influence of environmental factors on its deterioration.

Virtual Sensing of Key Variables in the Hydrogen Production Process: A Comparative Study of Data-Driven Models.

Hydrogen is an ideal energy carrier manufactured mainly by the natural gas steam reforming hydrogen production process. The concentrations of CH4, CO, CO2, and H2 in this process are key variables related to product quality, which thus need to be controlled accurately in real-time. However, conventional measurement methods for these concentrations suffer from significant delays or huge acquisition and upkeep costs. Virtual sensors effectively compensate for these shortcomings. Unfortunately, previously developed virtual sensors have not fully considered the complex characteristics of the hydrogen production process. Therefore, a virtual sensor model, called "moving window-based dynamic variational Bayesian principal component analysis (MW-DVBPCA)" is developed for key gas concentration estimation. The MW-DVBPCA considers complicated characteristics of the hydrogen production process, involving dynamics, time variations, and transportation delays. Specifically, the dynamics are modeled by the finite impulse response paradigm, the transportation delays are automatically determined using the differential evolution algorithm, and the time variations are captured by the moving window method. Moreover, a comparative study of data-driven virtual sensors is carried out, which is sporadically discussed in the literature. Meanwhile, the performance of the developed MW-DVBPCA is verified by the real-life natural gas steam reforming hydrogen production process.

Analytical comparison between batch and continuous direct compression processes for pharmaceutical manufacturing using an innovative UV-Vis reflectance method and chemometrics.

Advancements in industrial technologies and the application of quality by design (QbD) guidelines are shifting the attention of manufacturers towards innovative production techniques. In the pharmaceutical field, there is a significant focus on the implementation of continuous processes, in which the production stages are carried out continuously, without the need to interrupt the process and store the production intermediates, as in traditional batch production. Such innovative production techniques also require the development of proper analytical methods able to analyze the products in-line, while still being processed. The present study aims to compare a traditional batch manufacturing process with an alternative continuous one. To this end, a real pharmaceutical formulation was used, substituting the active pharmaceutical ingredient (API) with riboflavin, at the concentration of 2 %w/w. Moreover, a direct and non-destructive analytical method based on UV-Vis reflectance spectroscopy was applied for the quantification of riboflavin in the final tablets, and compared with a traditional absorbance analysis. Good results were obtained in the comparison of both the two manufacturing processes and the two analytical methods, with R2 higher than 0.9 for all the calculated calibration models and predicted riboflavin concentrations that never significantly overcame the 15 % limits recommended by the pharmacopeia. The continuous production method demonstrated to be as reliable as the batch one, allowing to save time and money in the production step. Moreover, UV-Vis reflectance was proved to be an interesting alternative to absorption spectroscopy, which, with the proper technology, could be implemented for in-line process control.

The impact of processing technology on microbial community composition and functional properties of Beninese maize ogi.

Traditionally fermented maize starch, called ogi, is produced to prepare akpan, a yoghurt-like street food widely consumed in Benin. Current maize ogi production practices were compared to assess the impact of different processing technologies on the characteristics of the fermented product as a basis to determine best practices. Maize starch slurry samples were collected from processors in five municipalities in southern Benin and analysed before fermentation (starch samples) and after spontaneous fermentation (ogi samples). Four technological pathways for maize starch production were distinguished based on variations in the duration of steeping the grains, which ranged from 6 to 72 h, and whether or not kneading of the wet flour before filtration was practised. Six categories of maize ogi were derived from the four technology groups based on the duration of the fermentation, which lasted from 6 to 24 h. The average pH of maize starch varied from 3.2 to 5.3, with the lowest values for the two technology groups that also had the highest lactate concentrations (9-11.8 g/L). The six maize ogi categories had a pH ranging from 3.1 to 4.0. Viable plate counts of lactic acid bacteria were similar for maize starch samples and for ogi samples, whereas yeast counts showed clear differences. Members of the genera Limosilactobacillus, Lactobacillus, Weissella, Streptococcus and Ligilactobacillus, dominated the bacterial community in maize starch, and were also dominant in maize ogi. The members of the genera dominating the fungal community in maize starch were also dominant in maize ogi, except for Aspergillus and Stenocarpella spp., which decreased in relative abundance by fermentation. The highest total free essential amino acid concentration was 61.6 mg/L in maize starch and 98.7 mg/L in ogi. The main volatile organic compounds in maize starch samples were alcohols, esters, and carboxylic acids, which also prevailed in maize ogi samples. The results indicate that the characteristics of traditional maize ogi depend on the processing technologies used to produce the maize starch before the intentional fermentation into ogi, with no clear-cut connection with the production practices due to high variations between samples from the same technology groups. This revealed the importance of a standardized maize starch production process, which would benefit controlling the starch fermentation and the characteristics of maize ogi. Further research is needed to understand the hidden fermentation during maize starch production for determination of the best practices that support the production of quality maize ogi.

The co-production process of an assessment programme: Between clarifying identity and developing the quality of French-speaking Belgian community health centres.

BACKGROUND: The assessment of primary care organizations is considered to be essential for improving care. However, the assessments' acceptability to professionals poses a challenge. Developing assessment programmes in collaboration with the end-users is a strategy that is widely encouraged to make interventions better targeted. By doing so, it can help to prevent resistance and encourage adherence to the assessment. This process, however, is rarely reported. This paper aims to fill this gap by describing the process of the co-production of an assessment programme for community health centres (CHCs) affiliated to the Federation of Community Health Centres (FCHC) in French-speaking Belgium. METHODS: We conducted a documentary study on the co-production of the assessment programme before carrying out semi-structured interviews with the stakeholders involved in its development. RESULTS: CHCs in French-speaking Belgium are increasing in number and are becoming more diverse. For the FCHC, this growth and diversification pose challenges for the meaning of CHC (an identity challenge) and what beneficiaries can expect in terms of the quality of organizations declaring themselves CHC (a quality challenge). Faced with this double challenge, the FCHC decided to develop an assessment programme, initially called Label, using participatory action research. During the co-production process, this initial programme version was abandoned in favour of a new name "DEQuaP". This new name embodies new objectives and new design regarding the assessment programme. When studying the co-production process, we attributed these changes to two controversies. The first concerns how much and which type of variety is desired among CHCs part of the FCHC. The second concerns the organization of the FCHC in its capacity as a federation. It shed light on tensions between two professional segments that, in this paper, we called "political professionalism" and "pragmatic professionalism". CONCLUSIONS: These controversies show the importance of underlying challenges behind the development of an assessment programme for CHCs. This provided information about the evolution of the identity of multidisciplinary organizations in primary care. Issues raised in the development of this assessment programme also show the importance of considering assessment methods that reflect and embody the current realities of these organizations and the way of developing these assessment methods.

A highly integrated digital PCR system with on-chip heating for accurate DNA quantitative analysis.

Digital polymerase chain reaction (dPCR) is extensively used for highly sensitive disease diagnosis due to its single-molecule detection ability. However, current dPCR systems require intricate DNA sample distribution, rely on cumbersome external heaters, and exhibit sluggish thermal cycling, hampering efficiency and speed of the dPCR process. Herein, we presented the development of a microwell array based dPCR system featuring an integrated self-heating dPCR chip. By utilizing hydrodynamic and electrothermal simulations, the chip's structure is optimized, resulting in improved partitioning within microwells and uniform thermal distribution. Through strategic hydrophilic/hydrophobic modifications on the chip's surface, we effectively secured the compartmentalization of sample within the microwells by employing an overlaying oil phase, which renders homogeneity and independence of samples in the microwells. To achieve precise, stable, uniform, and rapid self-heating of the chip, the ITO heating layer and the temperature control algorithm are deliberately designed. With a capacity of 22,500 microwells that can be easily expanded, the system successfully quantified EGFR plasmid solutions, exhibiting a dynamic linear range of 105 and a detection limit of 10 copies per reaction. To further validate its performance, we employed the dPCR platform for quantitative detection of BCR-ABL1 mutation gene fragments, where its performance was compared against the QuantStudio 3D, and the self-heating dPCR system demonstrated similar analytical accuracy to the commercial dPCR system. Notably, the individual chip is produced on a semiconductor manufacturing line, benefiting from mass production capabilities, so the chips are cost-effective and conducive to widespread adoption and accessibility.

Hepatotoxicity due to dietary supplements: state-of-the-art, gaps and perspectives.

Food supplements are products intended to complement the normal diet and consist of concentrated sources of nutrients or other substances with a nutritional or physiological effect. Although they are generally considered safe if the manufacturer's recommendations are followed, many of them have shown hepatotoxic properties. This can cause many diseases (e.g. steatohepatitis and cirrhosis) characterized by progressive damage and malfunction of the liver that in the long term can lead to death. A review of the literature was carried out to elucidate which dietary supplements have been associated with cases of hepatotoxicity in recent years, with emphasis on those relevant to the consumer and the new trends (e.g. cannabidiol). It has been reported that the supplements described as hepatotoxic are mainly of botanical origin (e.g. green tea or turmeric) and those used in sports (mainly anabolic androgenic steroids). There is a great variability of compounds described as causing liver damage, although sometimes it is not possible to identify them, because they are contaminants or adulterants of the products. In addition, the prevalence of toxic effects after the administration of supplements is difficult to define due to underreporting and the lack of specific studies. Globally regarding hepatotoxicity of dietary supplements, there is a paucity of well-conducted clinical trials on the efficacy of these compounds and the frequency of related liver damage, as the use of these products is largely uncontrolled.

C3PO: a crop planning and production process ontology and knowledge graph.

Vegetable crop farmers diversify their production by growing a range of crops during the season on the same plot. Crop diversification and rotation enables farmers to increase their income and crop yields while enhancing their farm sustainability against climatic events and pest attacks. Farmers must plan their agricultural work per year and over successive years. Planning decisions are made on the basis of their experience regarding previous plans. For the purpose of assisting farmers in planning decisions and monitoring, we developed the Crop Planning and Production Process Ontology (C3PO), i.e., a representation of agricultural knowledge and data for diversified crop production. C3PO is composed of eight modules to capture all crop production dimensions and complexity for representing farming practices and constraints. It encodes agricultural processes and farm plot organization and captures common agricultural knowledge. C3PO introduces a representation of technical itineraries, i.e., sequences of technical farming tasks to grow vegetables, from soil identification and seed selection to harvest and storage. C3PO is the backbone of a knowledge graph which aggregates data from heterogeneous related semantic resources, e.g., organism taxonomies, chemicals, reference crop listings, or development stages. C3PO and its knowledge graph are used by the Elzeard enterprise to develop knowledge-based decision support systems for farmers. This article describes how we built C3PO and its knowledge graph-which are both publicly available-and briefly outlines their applications.

Product safety aspects of plant molecular farming.

Plant molecular farming (PMF) has been promoted since the 1990s as a rapid, cost-effective and (most of all) safe alternative to the cultivation of bacteria or animal cells for the production of biopharmaceutical proteins. Numerous plant species have been investigated for the production of a broad range of protein-based drug candidates. The inherent safety of these products is frequently highlighted as an advantage of PMF because plant viruses do not replicate in humans and vice versa. However, a more nuanced analysis of this principle is required when considering other pathogens because toxic compounds pose a risk even in the absence of replication. Similarly, it is necessary to assess the risks associated with the host system (e.g., the presence of toxic secondary metabolites) and the production approach (e.g., transient expression based on bacterial infiltration substantially increases the endotoxin load). This review considers the most relevant host systems in terms of their toxicity profile, including the presence of secondary metabolites, and the risks arising from the persistence of these substances after downstream processing and product purification. Similarly, we discuss a range of plant pathogens and disease vectors that can influence product safety, for example, due to the release of toxins. The ability of downstream unit operations to remove contaminants and process-related toxic impurities such as endotoxins is also addressed. This overview of plant-based production, focusing on product safety aspects, provides recommendations that will allow stakeholders to choose the most appropriate strategies for process development.

Investigate the anaerobic degradation of high-acetone latex wastewater with magnetite supplement.

This study evaluated the effects of acetone on the anaerobic degradation of synthetic latex wastewater, which was simulated from the wastewater of the deproteinized natural rubber production process, including latex, acetate, propionate, and acetone as the main carbon sources, at a batch scale in 5 cycles of a total of 60 days. Fe3O4 was applied to accelerate the treatment performance from cycle 3. Acetone was added in concentration ranges of 0%, 0.05%, 0.1%, 0.15%-included latex, and 0.15%-free latex (w/v). In the Fe3O4-free cycles, for latex-added vials, soluble chemical oxygen demand (sCOD) was removed at 43.20%, 43.20%, and 12.65%, corresponding to the input acetone concentrations varying from 0.05% to 0.15%, indicating the interference of acetone for COD reduction. After adding Fe3O4, all flasks reported a significant increase in COD removal efficiency, especially for acetone-only and latex-only vials, from 36.9% to 14.30%-42.95% and 83.20%, respectively. Other highlighted results of COD balance showed that Fe3O4 involvement improved the degradation process of acetate, propionate, acetone, and the other COD parts, including the intermediate products of latex reduction. Besides, during the whole batch process, the order of reduction priority of the carbon sources in the synthetic wastewater was acetate, propionate and acetone. We also found that the acetate concentration appeared to be strongly related to reducing other carbon sources in natural rubber wastewater. Microbial community analysis revealed that protein-degrading bacteria Bacteroidetes vadinHA17 and Proteinniphilum and methylotrophic methanogens might play key roles in treating simulated deproteinized-natural-rubber wastewater.

Microbial Fermentation Processes of Lactic Acid: Challenges, Solutions, and Future Prospects.

The demand for lactic acid and lactic acid-derived products in the food, pharmaceutical, and cosmetic industries is increasing year by year. In recent decades, the synthesis of lactic acid by microbials has gained much attention from scientists due to the superior optical purity of the product, its low production costs, and its higher production efficiency compared to chemical synthesis. Microbial fermentation involves the selection of feedstock, strains, and fermentation modes. Each step can potentially affect the yield and purity of the final product. Therefore, there are still many critical challenges in lactic acid production. The costs of feedstocks and energy; the inhibition of substrates and end-product; the sensitivity to the inhibitory compounds released during pretreatment; and the lower optical purity are the main obstacles hindering the fermentation of lactic acid. This review highlights the limitations and challenges of applying microbial fermentation in lactic acid production. In addition, corresponding solutions to these difficulties are summarized in order to provide some guidance for the industrial production of lactic acid.

[Patent application of intelligent quality control technology in traditional Chinese medicine production process: a review].

In the new stage for intelligent manufacturing of traditional Chinese medicine(TCM) from pilot demonstration to in-depth application and comprehensive promotion, how to raise the degree of intelligence for the process quality control system has become the bottleneck of the development of TCM production process control technology. This article has sorted out 226 TCM intelligent manufacturing projects that have been approved by the national and provincial governments since the implementation of the "Made in China 2025" plan and 145 related pharmaceutical enterprises. Then, the patents applied by these pharmaceutical enterprises were thoroughly retrieved, and 135 patents in terms of intelligent quality control technology in the production process were found. The technical details about intelligent quality control at both the unit levels such as cultivation, processing of crude herbs, preparation pretreatment, pharmaceutical preparations, and the production workshop level were reviewed from three aspects, i.e., intelligent quality sensing, intelligent process cognition, and intelligent process control. The results showed that intelligent quality control technologies have been preliminarily applied to the whole process of TCM production. The intelligence control of the extraction and concentration processes and the intelligent sensing of critical quality attributes are currently the focus of pharmaceutical enterprises. However, there is a lack of process cognitive patent technology for the TCM manufacturing process, which fails to meet the requirements of closed-loop integration of intelligent sensing and intelligent control technologies. It is suggested that in the future, with the help of artificial intelligence and machine learning methods, the process cognitive bottleneck of TCM production can be overcome, and the holistic quality formation mechanisms of TCM products can be elucidated. Moreover, key technologies for system integration and intelligent equipment are expected to be innovated and accelerated to enhance the quality uniformity and manufacturing reliability of TCM.

Development and Innovation in Cooked Ham Produced in Spain.

The production of cooked ham has been gaining popularity in recent years in Spain. In general, the production process carried out by the companies remains traditional, and different production methods are therefore being sought to innovate and improve the quality of the product. This is either through pig crossbreeding, varying additives and ingredients, improving some stages of the production process, or providing nutritional and health claims that are useful to guiding the purchasing decision of consumers. Obviously, this series of changes must be subject to Spanish and European regulations in order to be marketed inside and outside the country.

The use of predictive models to develop chromatography-based purification processes.

Chromatography is the workhorse of biopharmaceutical downstream processing because it can selectively enrich a target product while removing impurities from complex feed streams. This is achieved by exploiting differences in molecular properties, such as size, charge and hydrophobicity (alone or in different combinations). Accordingly, many parameters must be tested during process development in order to maximize product purity and recovery, including resin and ligand types, conductivity, pH, gradient profiles, and the sequence of separation operations. The number of possible experimental conditions quickly becomes unmanageable. Although the range of suitable conditions can be narrowed based on experience, the time and cost of the work remain high even when using high-throughput laboratory automation. In contrast, chromatography modeling using inexpensive, parallelized computer hardware can provide expert knowledge, predicting conditions that achieve high purity and efficient recovery. The prediction of suitable conditions in silico reduces the number of empirical tests required and provides in-depth process understanding, which is recommended by regulatory authorities. In this article, we discuss the benefits and specific challenges of chromatography modeling. We describe the experimental characterization of chromatography devices and settings prior to modeling, such as the determination of column porosity. We also consider the challenges that must be overcome when models are set up and calibrated, including the cross-validation and verification of data-driven and hybrid (combined data-driven and mechanistic) models. This review will therefore support researchers intending to establish a chromatography modeling workflow in their laboratory.

Additive Manufacturing of Spinal Braces: Evaluation of Production Process and Postural Stability in Patients with Scoliosis.

Spinal orthoses produced using additive manufacturing show great potential for obtaining patient-specific solutions in clinical applications, reducing manual operations, time consumption, and material waste. This study was conducted to evaluate the production process of spinal orthoses produced by additive manufacturing, and to test the effects of 3D-printed braces on postural stability in patients with adolescent idiopathic scoliosis and osteogenesis imperfecta. Ten patients were recruited consecutively and were asked to wear a spinal orthosis produced by additive manufacturing for 2 weeks. The four phases of the production process for each brace were evaluated separately on a scale from 0 (not acceptable) to 3 (optimal). Postural stability in the unbraced and the two braced conditions (3D-printed and conventional) was assessed using validated metrics obtained from a wearable inertial sensor. The production process was evaluated as good in four cases, acceptable in five cases, and not acceptable in one case, due to problems in the printing phase. No statistically significant differences were observed in any of the postural balance metrics between the 3D-printed and conventional brace. On the other hand, postural balance metrics improved significantly with both types of braces with respect to the unbraced condition. Spinal orthoses produced with an innovative production process based on digital scans, CAD, and 3D printing are valid alternatives to conventionally produced orthoses, providing equivalent postural stability.

[Advantages and disadvantages of production quality of Fufang Danshen Prescription based on high-quality Chinese patent medicine evaluation criteria].

The big brand of Chinese patent medicine, Fufang Danshen Prescription(FDP), effective in promoting blood circulation, resolving blood stasis, regulating qi, and relieving pain, is wide in clinical application and diverse in dosage forms and products, but its quality and price are uneven, which causes problems for doctors and patients. To clarify the key links and weakness of quality control leading to the quality difference of FDP products, the present study carried out a comprehensive evaluation of the whole production cycle of FDP based on the "high-quality Chinese patent medicine evaluation criteria" and analyzed the advantages and disadvantages of production and quality of different FDP products according to scores. The results showed that the scores of various products in the "raw materials selection" varied greatly. The highest score(S1) and the lowest score(S2) differed by more than 3 times, indicating that different manufacturers had inconsistent requirements for the selection of raw materials, leading to fundamental differences in the quality of raw materials. The scores in the "production process" varied slightly, with an average score of 66.8%. The manufacturer S8 obtained the highest score(84.0%), which indicated the emergence of intelligent manufacturing production. The scores(with the average score of 44.0%) in the "quality control" were lower than those of the previous two items, which was attributed to the fact that most FDP products only met the "qualified" benchmark required by the 2020 edition of Chinese Pharmacopoeia, and their consistency and high quality were both uncontrollable. The scores in the "post-marketing research" were the lowest(with an average score of 28.5%), and most manufacturers were scored 0, which reflected little attention paid. Only a few brand manufacturers were scored acceptably and they were willing to carry out relevant research on post-marketing evaluation. The evaluation results demonstrated the key links and weakness leading to the production and quality differences of FDP from different manufacturers. It is expected to improve the quality of FDP, promote the formation of the "high quality and good price" mechanism, and provide information for the centralized procurement of governments.

Heterogeneous Variations on Historical and Future Trends of CO2 and Multiple Air Pollutants from the Cement Production Process in China: Emission Inventory, Spatial-Temporal Characteristics, and Scenario Projections.

Cement production is a major contributor to carbon dioxide (CO2) and multiple hazardous air pollutant (HAP) emissions, threatening climate mitigation and urban/regional air quality improvement. In this study, we established a comprehensive emission inventory by coupling the unit-based bottom-up and mass balance methods, revealing that emissions of most HAPs have been remarkably controlled. However, an increasing 6.0% of atmospheric mercury emissions, as well as 14.1 and 23.7% of fuel-related and process-related CO2 emission growth were witnessed unexpectedly. Industrial adjustment policies have imposed a great impact on the spatiotemporal changes in emission characteristics. Monthly emissions of CO2 and multiple HAPs decreased from December to February due to the "staggered peak production," especially in northern China after implementing the intensified action plan for air pollution control in winter. Upgrading environmental technologies and adjusting capacity structures are identified as dominant driving forces for reducing HAP emissions. Besides, energy intensity improvement can help offset some of the impact caused by the increase in clinker/cement production. Furthermore, scenario analysis results show that ultra-low emission and low-carbon technology transformation constitute the keys to achieve the synergic reduction of CO2 and multiple HAP emissions in the future.

Investigation on the microbiological hazards in an artisanal soft cheese produced in northern Italy and its production environment in different seasonal periods.

The present study aimed at assessing the occurrence of microbiological hazards (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Escherichia coli O157) in an artisanal soft cheese produced in northern Italy. In the same product total bacterial count, lactic acid bacteria and Enterobacteriaceae were enumerated, and pH and water activity measured in two batches sampled in summer and winter. Samples of raw materials, environmental swabs from the production processes and cheese during 15 days of storage at 2 and 8°C as well as dynamic temperature of 2°C for 5 days and 8°C for 10 days were collected and tested. The load of total bacterial count was significantly higher in the winter batch in comparison to the summer one, with a significant increase at the end of the storage period also noticed for lactic acid bacteria. Statistical higher values of pH were registered in raw materials and end of storage in winter batch. S. aureus was confirmed only in the winter batch within samples (n=4) of stored cheese. On plates used for E. coli O157 detection, colonies of Klebsiella pneumoniae and Klebsiella oxytoca were isolated. The results suggest that the highest bacterial population in the winter batch was associated to a higher pH in stored cheese and a higher number of biological hazards identified. Their isolation started in the maturation room suggesting this step as relevant for possible cheese contamination.

Increase recombinant antibody yields through optimizing vector design and production process in CHO cells.

Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. KEY POINTS: • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.

Retrospective Analysis of Autologous Chondrocyte-Based Cytotherapy Production for Clinical Use: GMP Process-Based Manufacturing Optimization in a Swiss University Hospital.

Cultured autologous human articular chondrocyte (HAC) implantation has been extensively investigated for safe and effective promotion of structural and functional restoration of knee cartilage lesions. HAC-based cytotherapeutic products for clinical use must be manufactured under an appropriate quality assurance system and follow good manufacturing practices (GMP). A prospective clinical trial is ongoing in the Lausanne University Hospital, where the HAC manufacturing processes have been implemented internally. Following laboratory development and in-house GMP transposition of HAC cell therapy manufacturing, a total of 47 patients have been treated to date. The main aim of the present study was to retrospectively analyze the available manufacturing records of the produced HAC-based cytotherapeutic products, outlining the inter-individual variability existing among the 47 patients regarding standardized transplant product preparation. These data were used to ameliorate and to ensure the continued high quality of cytotherapeutic care in view of further clinical investigations, based on the synthetic analyses of existing GMP records. Therefore, a renewed risk analysis-based process definition was performed, with specific focus set on process parameters, controls, targets, and acceptance criteria. Overall, high importance of the interdisciplinary collaboration and of the manufacturing process robustness was underlined, considering the high variability (i.e., quantitative, functional) existing between the treated patients and between the derived primary HAC cell types.